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Effect of antisense oligonucleotides for tissue factor on hepatic ischemia-reperfusion injury in the rat
HEPATIC
Effect of Antisense Oligonucleotides for Tissue Factor on Hepatic Ischemia-Reperfusion Injury in the Rat K. Nakamura, M. Okamoto, K. Akioka, M. Matsuyama, R. Yoshimura, H. Ushigome, Y. Kadotani, Y. Ohmori, and N. Yoshimura
N
OT ONLY with regard to hepatic failure, but also hepatectomy and liver transplantation, many physicians and surgeons have acknowledged ischemia-reperfusion injury as a serious clinical problem. Recent studies of ischemia-reperfusion injury have indicated that it may be related to the function of neutrophils, the action mechanism of inflammatory cytokines, and oxygen free radicals1,2; however, few reports have referred to the coagulation system. Tissue factor (TF) is an initiation factor for blood coagulation, and its expression is induced on macrophages and endothelial cells during inflammatory or immune responses. In our previous studies using the rat model, we reported the significance of TF and the effect of tissue factor pathway inhibitor (TFPI) in hepatic ischemic reperfusion injury.3,4 In this study, we exam-
Fig 1. Survival rates over a 5-day period after an ischemia time of 180 minutes. Six of 7 rats in the AS/TF-injected group survived, whereas 1 of 10 rats survived in the untreated ischemia-reperfused group.
ine an antisense phosphorothioate oligodeoxynucleotide to for specific inhibition of expression of rat TF, and assess the effect of the antisense oligodeoxynucleotide (ODN) on hepatic ischemic reperfusion injury in the rat. MATERIALS AND METHODS Phosphorothioate antisense ODN was produced as described elsewhere.5 Briefly, phosphorothioate ODNs were synthesized at the 1-mmol scale (Model 8900, Expedite Nucleic Acid Synthesis, PerSeptive Biosystems, Inc, Framingham, Mass) using modified phosphoramidite chemistries with -cyanoethoxyphosphoramidites (Glen Research, Sterling, Va). Oligos were purified by column chromatography using a Millipore HC18-HA column. The purity of the material was assessed by capillary electrophoresis, anion exchange, high-performance liquid chromatography, mass spectroscopy, and nuclear magnetic resonance imaging.6 The sequence of oligos was (antisense/TF [AS/TF]): 5⬘-CATGGGGATAGCCAT-3⬘. AS/TF was delivered intravenously to assess the expression of TF on endothelial cells of the rat liver. After 10 hours, by laparotomy of the Lewis rat, branches of the hepatic artery and portal vein leading to the median, left, and caudate lobes of the liver were clamped. The liver was reperfused after 180 minutes of ischemia. Rats were killed at 5 and 12 hours after reperfusion, and livers were harvested. TF expression was studied using immunohistochemical staining with monoclonal antibody (HTF-K108). Results are shown as mean ⫾ SD. The survival rate was From the Department of Organ Transplant and Endocrine Surgery, Kyoto Prefectural University of Medicine, Kyoto City, Kyoto, Japan (K.N., M.O., K.A., H.U., Y.K., Y.O., N.Y.); and Department of Urology, Osaka City University, Osaka, Japan (M.M., R.Y.). Supported in part by a Grant-in-Aid for Research on Human Genome, Tissue Engineering Food Biotechnology; Health Science Research Grants; and the Ministry of Health, Labour, and Welfare of Japan. Address reprint requests to Dr K. Nakamura, Department of Organ and Endocrine Surgery, Kyoto Prefectural University of Medicine, Kyoto City, Kyoto 602-8566, Japan.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02512-X
Transplantation Proceedings, 33, 3707–3708 (2001)
3707
3708
NAKAMURA, OKAMOTO, AKIOKA ET AL
Fig 2. Microscopic findings in the reperfused liver treated with AS/TF. With AS/TF treatment, focal necrotic areas were scarcely found at 5 (A) (original magnification ⫻200) and 12 hours (B) (original magnification ⫻200) after reperfusion by HE staining. With immunohistochemical staining, both at 5 hours (C) (original magnification ⫻200) and 12 hours (D) (original magnification ⫻200), TF staining was scarcely found on Kupffer cells and endothelial cells. calculated with the Kaplan–Meier method. Other statistical procedures were performed using the Friedman method.
RESULTS
Survival rates over a 5-day period were examined after an ischemic time of 180 minutes. Only 1 (10%) of 10 rats in the 180-minute ischemia group (n ⫽ 10) survived. However, by treatment with AS/TF, 6 of 7 rats in the 180-minute ischemia group survived (n ⫽ 7) (Fig 1). The ischemia-reperfused rat livers injected with AS/TF were stained with hematoxylin-eosin (HE) and monoclonal antibody to TF. With HE staining, some focal necrosis was found at 5 hours after reperfusion (Fig 2A), and slight focal necroses developed at 12 hours (Fig 2B). With immunohistochemical staining, both at 5 hours (Fig 2C) and 12 hours (Fig 2D), TF staining was found on Kupffer cells and endothelial cells, but the number of cells was low. DISCUSSION
It is known that TF assumes a significant role in cellular immune reactions, and is connected with the appearance of disseminated intravenous coagulation (DIC) and sepsis. In previous studies, we reported that the change of TF expression in the liver with ischemic reperfusion may initiate a coagulation mechanism at the level of the microcirculation, resulting in microcirculatory incompetence and thus making microthrombi, which, in turn, develop into discrete necrosis in the same ischemia-reperfused rat model. Using the same model, we demonstrated that the inhibition of TF expression upon administration of TFPI, which plays a dominant role in inhibiting extrinsic blood coagulation and begins by exposure of blood to TF at an injury site and formation of the complex between TF and plasma factor VII/VIIa, could have reduced the area of focal necrosis of
the reperfused rat liver and may have improved the survival rate in comparison with the nonadministered rats. We believe that inhibition of TF expression may be a significant factor in the prevention of organ viability in ischemia-reperfusion injury, and TFPI may preserve organ viability by the inhibition of the blood coagulation system. We designed synthetic antisense ODNs for TF, which were targeted to the 5⬘ untranslated region of rat TF mRNA, and assessed the effects of antisense ODNs in ischemia-reperfusion injury in rat livers. In this study, we chose the antisense method for the inhibition of TF expression. Synthetic phosphorothioate antisense ODN may prolong organ allograft survival by blocking the expression of specific targeted proteins or mRNA, by arrest of translation, inhibition of processing, or degradation of the targeted RNA.7 The use of the antisense method or TFPI, or combined use, might act to prevent organ damage due to ischemia-reperfusion injury. These results suggest that AS/TF may strongly suppress the expression of TF protein, and thereby inhibit ischemiareperfusion injury in the rat liver. REFERENCES 1. Colletti ML, Remick GD, Burtch DG, et al: J Clin Invest 85:1936, 1990 2. Jaeschke H, Farhood A, Smith WC: FASEB J 4:3355, 1990 3. Kobayashi Y, Yoshimura N, Nakamura K, et al: Transplantation 66:708, 1998 4. Yoshimura N, Kobayashi Y, Nakamura K, et al: Transplantation 67:45, 1999 5. Bennet CF, Condon T, Grimm T, et al: J Immunol 152:3530, 1994 6. Stepkowski SM, Wang ME, Condon TP, et al: Transplantation 66:699, 1998 7. Crooke ST: Annu Rev Pharmacol Toxicol 32:329, 1992
Effect of Antisense Oligonucleotides for Tissue Factor on Hepatic Ischemia-Reperfusion Injury in the Rat K. Nakamura, M. Okamoto, K. Akioka, M. Matsuyama, R. Yoshimura, H. Ushigome, Y. Kadotani, Y. Ohmori, and N. Yoshimura
N
OT ONLY with regard to hepatic failure, but also hepatectomy and liver transplantation, many physicians and surgeons have acknowledged ischemia-reperfusion injury as a serious clinical problem. Recent studies of ischemia-reperfusion injury have indicated that it may be related to the function of neutrophils, the action mechanism of inflammatory cytokines, and oxygen free radicals1,2; however, few reports have referred to the coagulation system. Tissue factor (TF) is an initiation factor for blood coagulation, and its expression is induced on macrophages and endothelial cells during inflammatory or immune responses. In our previous studies using the rat model, we reported the significance of TF and the effect of tissue factor pathway inhibitor (TFPI) in hepatic ischemic reperfusion injury.3,4 In this study, we exam-
Fig 1. Survival rates over a 5-day period after an ischemia time of 180 minutes. Six of 7 rats in the AS/TF-injected group survived, whereas 1 of 10 rats survived in the untreated ischemia-reperfused group.
ine an antisense phosphorothioate oligodeoxynucleotide to for specific inhibition of expression of rat TF, and assess the effect of the antisense oligodeoxynucleotide (ODN) on hepatic ischemic reperfusion injury in the rat. MATERIALS AND METHODS Phosphorothioate antisense ODN was produced as described elsewhere.5 Briefly, phosphorothioate ODNs were synthesized at the 1-mmol scale (Model 8900, Expedite Nucleic Acid Synthesis, PerSeptive Biosystems, Inc, Framingham, Mass) using modified phosphoramidite chemistries with -cyanoethoxyphosphoramidites (Glen Research, Sterling, Va). Oligos were purified by column chromatography using a Millipore HC18-HA column. The purity of the material was assessed by capillary electrophoresis, anion exchange, high-performance liquid chromatography, mass spectroscopy, and nuclear magnetic resonance imaging.6 The sequence of oligos was (antisense/TF [AS/TF]): 5⬘-CATGGGGATAGCCAT-3⬘. AS/TF was delivered intravenously to assess the expression of TF on endothelial cells of the rat liver. After 10 hours, by laparotomy of the Lewis rat, branches of the hepatic artery and portal vein leading to the median, left, and caudate lobes of the liver were clamped. The liver was reperfused after 180 minutes of ischemia. Rats were killed at 5 and 12 hours after reperfusion, and livers were harvested. TF expression was studied using immunohistochemical staining with monoclonal antibody (HTF-K108). Results are shown as mean ⫾ SD. The survival rate was From the Department of Organ Transplant and Endocrine Surgery, Kyoto Prefectural University of Medicine, Kyoto City, Kyoto, Japan (K.N., M.O., K.A., H.U., Y.K., Y.O., N.Y.); and Department of Urology, Osaka City University, Osaka, Japan (M.M., R.Y.). Supported in part by a Grant-in-Aid for Research on Human Genome, Tissue Engineering Food Biotechnology; Health Science Research Grants; and the Ministry of Health, Labour, and Welfare of Japan. Address reprint requests to Dr K. Nakamura, Department of Organ and Endocrine Surgery, Kyoto Prefectural University of Medicine, Kyoto City, Kyoto 602-8566, Japan.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02512-X
Transplantation Proceedings, 33, 3707–3708 (2001)
3707
3708
NAKAMURA, OKAMOTO, AKIOKA ET AL
Fig 2. Microscopic findings in the reperfused liver treated with AS/TF. With AS/TF treatment, focal necrotic areas were scarcely found at 5 (A) (original magnification ⫻200) and 12 hours (B) (original magnification ⫻200) after reperfusion by HE staining. With immunohistochemical staining, both at 5 hours (C) (original magnification ⫻200) and 12 hours (D) (original magnification ⫻200), TF staining was scarcely found on Kupffer cells and endothelial cells. calculated with the Kaplan–Meier method. Other statistical procedures were performed using the Friedman method.
RESULTS
Survival rates over a 5-day period were examined after an ischemic time of 180 minutes. Only 1 (10%) of 10 rats in the 180-minute ischemia group (n ⫽ 10) survived. However, by treatment with AS/TF, 6 of 7 rats in the 180-minute ischemia group survived (n ⫽ 7) (Fig 1). The ischemia-reperfused rat livers injected with AS/TF were stained with hematoxylin-eosin (HE) and monoclonal antibody to TF. With HE staining, some focal necrosis was found at 5 hours after reperfusion (Fig 2A), and slight focal necroses developed at 12 hours (Fig 2B). With immunohistochemical staining, both at 5 hours (Fig 2C) and 12 hours (Fig 2D), TF staining was found on Kupffer cells and endothelial cells, but the number of cells was low. DISCUSSION
It is known that TF assumes a significant role in cellular immune reactions, and is connected with the appearance of disseminated intravenous coagulation (DIC) and sepsis. In previous studies, we reported that the change of TF expression in the liver with ischemic reperfusion may initiate a coagulation mechanism at the level of the microcirculation, resulting in microcirculatory incompetence and thus making microthrombi, which, in turn, develop into discrete necrosis in the same ischemia-reperfused rat model. Using the same model, we demonstrated that the inhibition of TF expression upon administration of TFPI, which plays a dominant role in inhibiting extrinsic blood coagulation and begins by exposure of blood to TF at an injury site and formation of the complex between TF and plasma factor VII/VIIa, could have reduced the area of focal necrosis of
the reperfused rat liver and may have improved the survival rate in comparison with the nonadministered rats. We believe that inhibition of TF expression may be a significant factor in the prevention of organ viability in ischemia-reperfusion injury, and TFPI may preserve organ viability by the inhibition of the blood coagulation system. We designed synthetic antisense ODNs for TF, which were targeted to the 5⬘ untranslated region of rat TF mRNA, and assessed the effects of antisense ODNs in ischemia-reperfusion injury in rat livers. In this study, we chose the antisense method for the inhibition of TF expression. Synthetic phosphorothioate antisense ODN may prolong organ allograft survival by blocking the expression of specific targeted proteins or mRNA, by arrest of translation, inhibition of processing, or degradation of the targeted RNA.7 The use of the antisense method or TFPI, or combined use, might act to prevent organ damage due to ischemia-reperfusion injury. These results suggest that AS/TF may strongly suppress the expression of TF protein, and thereby inhibit ischemiareperfusion injury in the rat liver. REFERENCES 1. Colletti ML, Remick GD, Burtch DG, et al: J Clin Invest 85:1936, 1990 2. Jaeschke H, Farhood A, Smith WC: FASEB J 4:3355, 1990 3. Kobayashi Y, Yoshimura N, Nakamura K, et al: Transplantation 66:708, 1998 4. Yoshimura N, Kobayashi Y, Nakamura K, et al: Transplantation 67:45, 1999 5. Bennet CF, Condon T, Grimm T, et al: J Immunol 152:3530, 1994 6. Stepkowski SM, Wang ME, Condon TP, et al: Transplantation 66:699, 1998 7. Crooke ST: Annu Rev Pharmacol Toxicol 32:329, 1992